A structural, epidemiological & genetic overview of Klebsiella pneumoniae carbapenemases (KPCs).

Klebsiella pneumoniae carbapenemases (KPCs) are plasmid encoded carbapenem hydrolyzing enzymes which have the potential to spread widely through gene transfer. The instability of upstream region of blaKPC accelerates emergence of different isoforms. Routine antibiotic susceptibility testing failed to detect KPC producers and some commercial kits have been launched for early identification of KPC producers. Notable among the drugs under development against KPC are mostly derivatives of polymixin; ß-lactamase inhibitor NXL104 with combination of oxyimino cephalosporin as well as with ceftazidime; a novel tricyclic carbapenem, LK-157, potentially useful against class A and class C enzymes; BLI-489-a bicyclic penem derivative; PTK-0796, a tetracycline derivative and ACHN-490. Combination therapy might be preferable to control KPC infections in immediate future. Clinicians are likely to opt for unconventional combinations of antibiotics to treat KPC infections because of unavailability of alternative agents. The KPCs have become endemic in many countries but there is no optimal treatment recommendation available for bacteria expressing KPCs. Reports of outbreaks involving KPCs have focused mainly on laboratory identification, empirical treatment outcomes and molecular epidemiology. This review includes information on the emergence of KPC variants, limitations of phenotyping methods, available molecular methods for identification of the KPC variants and treatment options highlighting the drugs under development.

Introduction

Emergence of multi-drug resistance (MDR) among Enterobacteriaceae is a threat observed in the last two decades. The most important and common therapeutic challenge posed by Enterobacteriaceae is resistance to carbapenems. Carbapenems are broad spectrum antibiotics structurally very similar to penicillin but contain a sulphur group at C1 position. Several Gram-negative pathogens have become resistant to these by acquiring any one or more of the following mechanisms: structural alterations in drug targets like penicillin binding proteins (PBPs), porin loss, upregulation of efflux pumps and expression of carbapenemases1. One of the most commonly observed mechanism among Gram-negative pathogens is production of carbapenem inactivating enzymes, called carbapenemases2. All carbapenemases are β-lactamases but not all β-lactamases are carbapenemases. These periplasmic enzymes hydrolyze beta lactam antibiotics either by alteration in the target site of the antibiotic that reduces its binding capacity or modification of the antibiotic so that it is no longer recognized by the target. Carbapenemases are broadly divided into two major types based on the amino acid sequences: metallo β-lactamases (Class B) containing zinc at the active site whereas serine β-lactamases (Classes A, C and D) containing serine at the active site. The Klebsiella pneumonia carbapenemases (KPC) along with other members like SME, IMI, NMC, GES constitute the class A13. Among these, the carbapenemase is more important than others due to its prevalence and transmissibility through plasmids4. Class C enzymes are chromosomally encoded, which were mostly found in several Gram-negative pathogens such as Acinetobacter spp., Aeromonas spp. and Enterobacter spp. The combination of class A and class C β-lactamases is highly potent as these confer resistance to most cephamycins, penicillins, cephalosporins and are not inhibited by clinically used inhibitors such as clavulanic acid5. Most common type of carbapenemases seen in Acinetobacter baumannii is oxacillanases. The class B enzymes include IMP, VIM, SPM, GIM, NDM and SIM families detected primarily in Pseudomonas aeruginosa1. This review provides information on one of the class A carbapenemase called KPC named after the organism in which it was first identified67. Later, it was detected in various other Gram-negative bacteria like Enterobacteriaceae (Escherichia coli, K. oxytoca, Enterobacter spp., Serratia spp., Salmonella spp., and Citrobacter freundii) and Pseudomonadaceae (Pseudomonas aeurignosa)289101112. Class A carbapenemase production has become a progressively common mechanism of resistance across the world313141516171819. The KPCs are encoded on a conjugative plasmid, carried in Tn3 (Tn4401) based transposon91920. Though class A enzymes and Bush's 2f type of hydrolases21 are comparable in many characteristics, the KPC enzymes have two unique features: (i) these are borne on transferable conjugative plasmids, and (ii) capable of hydrolyzing advanced derivatives of cephalosporins like cefotaxime122. Tazobactam and clavulanic acid are partial inhibitors of class A enzymes whereas boronic acid acts as complete inhibitor1923. Thirteen variants of KPC (blaKPC 1-13) have been identified till date, which differ from each other by non-synonymous mutations1224. These variants also show differences in their substrate (carbapenem) profile and affinity. So, there is a need for specific identification of these KPC variants in such Gram-negative bacteria (GNB) that would allow clinicians make early treatment decisions for nosocomial infections7.

Epidemiology

Initially KPC producing organisms were restricted to sporadic outbreaks, but the situation became difficult because of global explosion and endemicity in both developed and developing countries8182526. From 2001 till date, 13 KPC subtypes have been found among Gram-negative organisms in different geographical regions3410111619262728293031. Dissemination of variants occurs through acquisition of stable genetic elements by gene transfer mechanisms. Genotyping and sequence characterization of KPC by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST), conducted in different countries including India, have reported that all KPC expressing strains harbour a single complex clone C11 which further indicates the origin of KPC from the same323334. The major clone belongs to a sequence type called ST258 which is possibly responsible for all endemic outbreaks of KPC and also of international dissemination3. Other sequence types of KPC were also identified, for example, ST14 in Midwest United States6. Interestingly, ST11 is the predominant type of K. pneumonia associated with KPC production in China, which is closely related to ST258 and the latter is found more commonly elsewhere in the world2535. The overall mortality rate associated with KPC producing organisms is 50 per cent36. The awareness of the incidence of resistance to carbapenems from India is increasing3738394041. Gupta et al42 reported 17-22 per cent of hospital acquired infections among Gram-negative pathogens, while Wattal et al43 reported 13-51 per cent prevalence of carbapenem resistant Enterobacteriaceae (CRE).

Genetics of the KPC variants

Genes encoding versatile carbapenemases not only reside on chromosomes and but are also found on extrachromosomal DNA and transposons. Chromosomally encoded carbapenem inactivating enzymes evolved as defense mechanism in these bacteria also have a role in regulation of cell wall synthesis12244. The expression of these plasmid encoded enzymes gets upregulated by various interacting bacterial factors like (i) intrinsic hydrolytic activities shown by KPC variants, (ii) the genetic relatedness and control of carbapenemase production, and (iii) the presence or absence of other broad-spectrum β-lactamases and porin mutations145. It is necessary to establish how the bacterium is able to show resistance to all classes of antibiotics and the mutations that are responsible for resistance. The cause of rapid disseminatin of blaKPC is the presence of transposable element Tn4401 transposon which can integrate into various plasmids of non-clonally related organisms. Tn4401 is found at various loci on plasmids, which differ in size and incompatibility group. Tn4401 is approximately 10 kb in size characterized by the terminal two 39 bp imperfect inverted repeat sequences, harbours insertion sequences ISKpn6 and ISKpn7 and a set of site specific transposase and resolvase genes for insertion and excision of transposon946. It can integrate into different plasmids and evolve as isoforms. Three isoforms, a, b, and c of Tn4401, have been described by Naas et al9, differing by a 99-215 bp sequence upstream of blaKPC, while isoforms with 68 bp and 255 bp (Tn4401 e) deletions have also been reported46. A 5.3 kb region consisting of insertion sequence ISKpn7 and partial tnpA gene and more than half of the blaKPC gene has been deleted from the transposon Tn4401. This novel truncated transposon was named as Tn4401d by Chen et al46. Tn4401 can transpose with 5 bp target site duplication without any target site specificity (Fig. 1). Mobility and plasticity of Tn4401 lead to genetic diversity of plasmids and to the spread of KPC. Several studies have concluded81946 that blaKPC gene has variable upstream region due to heterogeneous genetic environment and conserved downstream region. Genetic environment of each variant shows that instability in the upstream region of blaKPC may be involved in variations of KPC. Several studies were conducted to elucidate the changes in non-conserved variable regions which were involved in the evolution of variants and the consequent changes in the spectrum of enzyme activity946. The enzyme activity decreases if the change is due to insertion sequences and increases if there are deletions19. Inconsistencies in detection of KPC variants may be due to acquisition of genes from other sources or due to indiscriminate mutational events. KPC genes are endowed with all molecular features such as transposon, self-transferable plasmids, competent Sequence Types (STs) which are responsible for their propagation between members of Enterobacteriaceae and other Gram-negative members. Phylogenetic tree for KPC subtypes was constructed (Fig. 2) which showed that most of the members were scattered throughout the phylogenetic tree and they were not grouped under single genetic lineage. This indicates that the distribution of KPC is through horizontal gene transfer (HGT) and not by clonal spread4447.

Fig. 1

Schematic diagram of Tn4401b and other isoforms on plasmids of Klebsiella pneumoniae isolates. IRR and IRL are inverted repeats (black triangles), flanked by 5 bp target site duplications (TSD). Insertion sequences: ISKpn6, ISKpn7; Transposase: tnpA, resolvase (tnpR). Dark triangles: deletions in isoforms a, c; Tn4401d truncated isoform with 5.3 kb deletion (flower bracket); Tn4401e: 255 bp deletion (entire putative promoter and transcription start site in upstream region91946.

Fig. 2

Circular phylogenetic tree of currently known KPC enzymes, showing common ancestry (KPC-2) of all other variants. Amino acid sequences of KPC enzymes (GenBank- http://www.ncbi.nlm.nih.gov) aligned with Clustal W (http://www.ebi.ac.uk/Tools/msa/clustalw2/) using default parameters. The phylogenetic tree was constructed using MEGA 5.16.0 and the NJ (Neighbor Joining) method and the vertical bar in between KPC-13 - KPC-4 indicates measure for amino acid sequence diversity.

Characteristics of KPC variants

Life threatening nosocomial infections are associated with these KPC producing Gram-negative pathogens. KPC-2 is considered as the ancestral variant and it is observed mostly among the clinical isolates11. All variants arose from KPC2, which differ from one another by one or two point mutations45. Mostly these changes have been noticed frequently at four conserved amino acid positions, namely nucleotides 147, 308, 716, and 814 which encode for different amino acids1112024. Nucleotide changes in KPC-2 in comparison with other variants are compiled with the help of EMBOOS tools (www.http://www.ebi.ac.uk/Tools/emboss/) (Table I). Single amino acid substitutions allow lactamases to increase their substrate profile and gradually alter the ability of lactamases to hydrolyze lactams before they reach their target sites (Table II). Structural analysis has revealed that the characteristic feature of class A enzymes is that they possess S-X-X-K, S-D-N and K-T-G motifs at positions 70, 130 and 234, respectively in the protein which are responsible for efficient carbapenem hydrolytic activity compared to other classes1. The bicine buffer molecule of KPC-2 interacts through its carboxyl group to conserved active site residue of K234. Within the class A group, KPC enzymes evolved through various modalities including variation in primary sequence, selective pressure by β-lactam antibiotics, mutation rates, changes in three dimensional structure and protein stability11149. Inhibitor profiling of KPC could help in accurate identification of the variants and hence an effective treatment strategy. Overall features of all KPC variants have been assembled and presented (Table III)1732.

Table I

Results of pair-wise alignment (Emboss) between KPC enzymes

Table II

Substrate profile for variants of blaKPC

Table III

Biochemical characteristics of KPC variants identified till date

Methods for KPC identification

The dissemination and emergence of KPC producers is a serious threat to public health because it drastically limits the treatment options2050. It not only confers resistance to carbapenems, but also to almost all other β-lactams. Production of these enzymes could be confirmed by several phenotypic tests (e.g. modified Hodge tests) whereas genotypic methods such as multiplex PCR are being used to conform blaKPC genes. Identifying KPCs is still a challenge for many clinical microbiology laboratories because bacteria expressing KPC do not always show high minimum inhibitory concentration (MICs), on the contrary, many isolates remain within susceptible or intermediate levels, frequently missed by technicians. Several methods have been developed so far for identification but each has its own limitations. A novel microtitre plate based chromogenic method called Carba NP has been developed and reported as one of the screening tests for carbapenemases5152. Several phenotypic and molecular methods have been introduced for KPC identification but phenotypic methods are known to be time consuming. Though automation has reduced the time to report and enabled easy handling of multiple samples simultaneously, many such systems frequently yield false positive/false negative results due to errors in identification of carbapenamase production53. For phenotypic identification of KPC, diffusion of meropenem disk with aminophenylboronic acid, dipicolinic acid and cloxacillin was developed54. Broth microdilution method (BMD) is one of the accurate and reliable methods for the identification of KPC-mediated carbapenem resistance5155. Among impienem, meropenem and ertapenem based automated systems like Microscan and Vitek-2, ertapenem based automated system proved to be more reliable and rapid for the detection of carbapenemases51. When compared with other phenotypic methods CHROMagar™ method showed 98 per cent efficiency in the detection of carbapenamases (KPC, VIM, NDM, etc.)11. In antimicrobial susceptibility tests higher MICs of the antibiotic used could be due to increased levels of expression of these enzymes and low penetration of drug into periplasmic space. In India, resistance to available carbapenems among K. pneumonia was reported as 43.6 per cent (meropenem), 32 per cent (imipenem), 20.3 per cent (ertapenem) and 60 per cent (colistin)2740. Ertapenem resistance has been shown to be more accurate for detection of KPC producers. This heterogeneous expression of carbapenemases by microorganisms makes it difficult to give uniform values for phenotypic methods53. Modified Hodge test (MHT) one of the phenotypic methods to identify carbapenemase producers among Enterobacteriaceae, demonstrated high levels of sensitivity and specificity for detection of KPC activity51. However, MHT is prone to give false positive results particularly with strains producing CTX-M extended-spectrum β-lactamases (ESBLs) and AmpC β-lactamase47495153. Hence, in geographical areas where ESBL is frequently reported, alternative methods are warranted. Melt curve analysis in real time PCR gives accurate results but requires high expertise and is also expensive2456. Though few commercial kits are available for the identification of KPC directly from the clinical samples, but discrepancies exist among these kits57585960. Real time PCR is being explored as a platform for rapid identification of blaKPC variants57585960.

Emerging treatment options and clinical outcomes

Nearly 89 per cent of nosocomial infections are caused mainly by K. pneumonia and its foremost site for infection is blood (52%), secondary respiratory related problems comprise 30 per cent, whereas urinary tract infections (UTI) contribute 10 per cent of infections in both acute care as well as tertiary care facilities37. Specific factors which play a role in pathogenesis have not yet been identified in KPC producing organisms. Major risk factors for infections are (i) long term hospital stay, (ii) mechanical ventilation, and (iii) previous antibiotic therapy and other factors20. Treatment failures occur due to intrinsic (genetic factors) and external factors interfering with proper identification of KPC gene. Resistance to other antibiotics includes fluoroquinolones, trimethoprim/sulphamethoxazole and aminoglycosides. Current options for treatment of KPC include tigecycline and colistin. However, each of these drugs and polymixin B as monotherapy results in frequent failures in treatment compared to combination therapy. Therapeutic options under development against KPC are mostly derivatives of polymixin (e.g. NAB739, NAB740); others include β-lactamase inhibitor NXL104 with combination of oxyimino cephalosporin as well as with ceftazidime461. A novel tricyclic carbapenem LK-157 has emerged as a potential drug against class A and class C enzymes62. BLI-489 is a bicyclic penem derivative which has shown inhibitory effect against several enzymes though has not been extensively tested on KPC producing microbes63. Two other antibiotic derivatives are under development against KPCs, one is a tetracycline derivative called PTK-0796 and other is aminoglycoside derivative ACHN-490 called neoglycoside46164. Clinicians are likely to opt for unconventional combinations of antibiotics to treat KPC infections because of unavailability of alternative agents. U.S. Food and Drug Administration (FDA) approved a promising agent Doribax (doripenem) which has broad spectrum activity65. In Greece, one cohort study reported that 88 per cent of the patients who underwent combination therapy, almost 22 per cent showed failures6. Improvements in combination therapy might be preferable for control of KPC infections for immediate future66.

Conclusion

KPC producing Enterobacteriaceae members have become a major concern causing serious health problems worldwide. Accumulation and transfer of KPC resistance determinants rapidly through horizontal gene transfer leads to an increase in mortality and morbidity. Variations in resistance determinants add to the complication in identification of blaKPC gene. The reduced susceptibility to carbapenems makes it necessary to add inhibitors to carbapenemases like clavulanic acid in the treatment regimen or include inhibitors like EDTA in culture characterization. Evaluation and characterization of Tn4401, plasmids and transposons may help in identification of natural reservoir for blaKPC which might facilitate to control the dissemination of resistance. Further research is required to understand the genetic relatedness among variants. There is also a need to explore the heterogeneity in the genetic environment of blaKPC and to implement accurate detection methods. It is essential that hospitals strictly administer screening and implement control measures for reducing emergence and spread of carbapenem resistant bacteria.